Recent studies have identified a novel pathway for increased risk for developing cardiovascular disease (CVD). People with high plasma concentrations of trimethylamine N-oxide (TMAO) are more likely to have increased CVD events in clinic-based samples. In spite of this striking association, there are limitations in current understanding, as we do not understand why some people are more likely to generate TMAO from dietary precursors (choline and carnitine) than are other people. Specifically, it is not known if (and how) common genetic variants and dietary choline/L-carnitine interact to affect TMAO levels or the mechanism by which TMAO increases CVD. Furthermore, many questions remain regarding the contribution of the microbiome to the TMAO-CVD pathway; for example we do not know which bacteria in the gut are regulating TMAO levels and if host genetics affects the amount of these bacterial species. We suggest that discovery of which genes are responsible for modulation of plasma TMAO concentrations and how these genes interact with gut microbiota is essential if we are to understand the association between TMAO and CVD risk. In the proposed work, we capitalize upon our expertise in mouse genetics to further understand the regulation of TMAO plasma concentrations. In particular we focus on genetic studies in hyperlipidemic mice that integrate microbiome analysis to better understand the genetic and microbial that may mediate both TMAO levels and cardiovascular risk. These studies allow us to leverage the well-controlled studies in mice using the most advanced mouse genetic resources available called the Diversity Outbred (DO) mice. Using a simple breeding scheme we will develop a panel of 600 hyperlipidemic DO mice for genetic mapping and use these for detailed analysis of the microbiome.